Myosin is a family of proteins that demonstrate force-generated ATPase activity when it interacts with actin. Myosin heavy chain (MHC) protein isoform diversity in vertebrates is generated by multiple genes as well as by alternative splicing of the pre-mRNA. Previous studies in this laboratory have demonstrated the existence of a neuron-specific nonmuscle MHC-B isoform, in addition to the ubiquitously expressed form of nonmuscle MHC-B. This neuron-specific isoform is generated by alternative splicing of a single cassette type of exon N30. Inclusion or exclusion of exon N30 is cell type-specific, with inclusion being restricted to neuronal cells and being regulated during cell differentiation. To understand the mechanism(s) of neuron-specific alternative splicing, we have been characterizing cis-elements which are required for neuron-specific recognition of the N30 exon using a minigene transfection system. Deletion and mutation analysis of the 3' intron region of N30 demonstrates that neuron-specific N30 exon splicing requires a distal intronic enhancer (DIE) sequence located 1.5 kb downstream from the N30 exon. Sequence analysis of the proximal region of the N30 exon reveals that there are two suboptimal sequences for recognition by basal splicesome components. The 5' splice donor site of the N30 exon is atypical. The N30 exon is followed by GC instead of the usual GT. The polypyrimidine tract preceding the 3' splice acceptor site of N30 is short and interrupted frequently by purines (suboptimal). When the 5' splice site of N30 is changed to an optimal site, an increase in N30 inclusion is seen in both neuronal and non-neuronal cells to some extent. But the dependency of cell type as well as the dependency of the DIE sequence for N30 recognition are still retained. In contrast, when the polypyrimidine tract of N30 is improved, almost 100% of the mRNA includes the N30 exon in both the presence or absence of the DIE sequence and regardless of cell type. Thus, the weak nature of the polypyrimidine tract preceding the N30 exon is a critical parameter for neuron-specific alternative splicing of the N30 exon.